Optically probing sub-diffractional biological features has always challenged researchers and clinicians. Remarkable efforts have been made to overcome this challenge from different aspects of physics. Near-field scanning optical microscopy (NSOM) collects evanescent electromagnetic waves and marked the first time that Abbe's diffraction limit was broken. Attempts to convert evanescent waves to propagating waves also led to the development of hyperlens and superlens. Stimulated emission depletion microscopy (STED), photoactivation localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) control the switchable fluorescent reporters to resolve sub-diffractional objects in the far field, while structured-illumination microscopy (SIM) extends the collection range of the spatial frequency to achieve high resolution. Confocal light absorption and scattering spectroscopy microscopy (CLASS) utilizes Mie scattering spectrum to resolve sub-wavelength organelles without exogenous labels. More recently, spatial light interference Microscopy (SLIM) based on wave interference images nanoscale cell topography due to intrinsic scattering. Other techniques based on this interference principle such as spectral-domain optical coherence phase microscopy (SD-OCPM) are also reported. However, the challenge remains within intact tissue since the above methods are only applicable to cells.
Many of the previous attempts have required fluorescent reporters and are not able to quantify tissue structures. Furthermore, current in vivo optical measurement modalities for biological tissues are extremely challenging, because the back reflectance scheme inhibits the ability to quantify full optical scattering properties for a given sample.
Optical Coherence Tomography (OCT) is a high-resolution medical and biological imaging technology. OCT is analogous to ultrasound B-mode imaging except reflections of low-coherence light are detected rather than sound. OCT detects changes in the backscattered amplitude and phase of light. This imaging technique is attractive for medical imaging because it permits the imaging of tissue microstructure in situ, yielding micron-scale imaging resolution without the need for excision and histological processing. Because OCT performs imaging using light, it has a one- to two-order-of-magnitude higher spatial resolution than ultrasound and does not require contact with tissue.
OCT is well known to provide depth-resolved images of tissue up to approximately 1 mm. Yet the spatial resolution of these images is fundamentally limited by a temporal coherence length, which is typically greater than 1 μm and, in commercial instruments, greater than about 10 μm.
Previous attempts in the art to quantify a complete measurement of all optical light scattering properties have failed. Thus, there is a need to provide methods that enable one to acquire the spatial information of OCT at the sensitivity of sub-micron, sub-resolution length scales of light scattering.